The general focus of this research is on understanding the coordination of the cellular and molecular events that must occur as proliferating cells transition to terminal differentiation. The retinoblastoma (pRb) and p300 protein families regulate various aspects of both tissue-specific gene expression and cell cycle control in most cell types. These proteins are bound and inactivated by the adenovirus 12S.E1A protein. We have generated MC3T3-E1 cell lines stably expressing either the wild-type 12S.E1A gene or mutants that selectively target either p300/CBP or pRb/p130/p107 and compromise their functions. These studies revealed that inactivation of these two families of proteins resulted in the suppression of two osteoblast marker genes: alkaline phosphatase and osteopontin. The unique dependence of ALP expression on both the pRb and p300 families implies a specific mechanistic link between ALP expression, cell cycle regulation, and terminal differentiation. Osteopontin, however, does not require the pRb and p300 family but instead is dependent on the expression of alkaline phosphatase and the subsequent inorganic phosphate produced by the enzyme in the extracellular environment. The discovery that inorganic phosphate in the medium results in the upregulation of osteopontin defines a novel and potentially important regulatory mechanism for gene regulation. Osteopontin is expressed in most tissues and therefore this novel mechanism has the potential to be globally important. Osteopontin protein levels are upregulated during cell injury and are associated with the pathology of many diseased tissues, including kidney stones, atherosclerosis, and transformed cells. Studies involving reduced levels of osteopontin have shown that it is required for wound healing and as a key cytokine required for efficient type-1 immune responses. It will be interesting to determine whether osteopontin is unique in its response to inorganic phosphate or whether there are other genes (promoters) that exhibit this sort of regulation. It will also be interesting to identify key components of the pathway required for inorganic phosphate regulation, including critical signal transducers and any phosphate-responsive elements in the osteopontin promoter. A related project in the lab revolves around the role of p270 in differentiation. p270 is an integral member of human SWI/SNF complexes, first identified through its shared antigenic specificity with p300 and CBP (Dallas PB, et al. Mol Cell Biol 18: 3596-3603, 1998). SWI/SNF complexes were discovered in yeast cells where they are involved in the regulation of multiple inducible genes, including those required for the mating-type switch and sucrose fermentation pathways. Although there appear to be multiple SWI/SNF-related complexes of variable composition in eukaryotic cells, all are able to alter chromatin structure through ATP-dependent mechanisms. These changes may subsequently permit the recruitment of other general and gene-specific activator molecules while facilitating the transcriptional process. A recently defined motif, LXXLL (L is leucine and X is any amino acid), has been demonstrated to be necessary and sufficient for interaction with liganded nuclear hormone receptors. The list of proteins containing this functional motif is growing, but to date it appears it is the hallmark of the protein family of transcriptional "co-activators". The most studied proteins of this group are p300 and CBP (CREB-binding protein). The function of these proteins is to assist in the formation of a complex between upstream transcriptional activators/enhancers and the basal transcriptional machinery, including TBP. p270 has a group of four LXXLL motifs located towards the C-terminus of the protein. The potential interaction between various nuclear hormone receptors and p270, a protein associated with a complex that contains chromatin-modifying activity, is very exciting. It suggests a role for p270 as an integrator of hormone signaling and transcription initiation.